1. Field of the Invention
The present invention relates, in general, to a kinase which in its activated state is capable of site-specific phosphorylation of IxcexaBxcex1, IxcexaBxcex1 kinase. In particular, the present invention relates to the purified kinase, purified polypeptide subunits of the kinase, nucleic acid molecules coding for the purified polypeptide subunits; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to the kinase or its polypeptide subunits; hybridomas containing the antibodies; nucleic acid probes for the detection of the nucleic acid encoding the kinase; a method of detecting nucleic acids encoding the kinase or polypeptides of the kinase in a sample; and kits containing nucleic acid probes or antibodies. This invention further relates to bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess, or prognose a mammal afflicted with an undesired activation of NF-xcexaB. This invention also relates to ligands, agonists, and antagonists of the kinase, and diagnostic and therapeutic uses thereof. This invention also relates to bioassays using the kinase or polypeptides of the kinase of this invention to identify ligands, agonists, and antagonists. More specifically, this invention relates to selective inhibitors of the kinase and to structure-based design of ligands, agonists, and antagonists of the kinase. This invention further relates to gene therapy using the nucleic acids of the invention.
2. Related Art
Regulation of the immune and inflammatory responses requires the activation of specific sets of genes by a variety of extracellular signals. These signals include mitogens (e.g., LPS and PMA), cytokines (e.g., TNF-xcex1 and IL-1xcex2), viral proteins (e.g., HTLV-1 Tax), antigens, phosphatase inhibitors (e.g., okadaic acid and calyculin A), and UV light. The rel/NF-xcexaB family of transcriptional activator proteins plays an essential role in the signal transduction pathways that link these signals to gene activation (reviewed by Siebenlist, U. et al., Annu. Rev. Cell. Biol. 10:405-455 (1994); Baerle and Henkel, Annu. Rev. Immunol. 12:141-179 (1994); Thanos and Maniatis, Cell 80:529-532 (1995); Finco and Baldwin, J. Biol. Chem. 24:17676-17679 (1993); Verma, I. M. et al., Genes and Dev. 9:2723-2735 (1995)). NF-xcexaB (p50/RelA(p65)), and other heterodimeric rel family proteins are sequestered in the cytoplasm through their association with IxcexaBxcex1 or IxcexaBxcex2, members of the IxcexaB family of inhibitor proteins. In the case of IxcexaBxcex1, and most likely IxcexaBxcex2, stimulation of cells leads to rapid phosphorylation and degradation of the inhibitor. Consequently NF-xcexaB is released and translocates into the nucleus where it activates the expression of target genes. Phosphorylation of IxcexaBxcex1 per se is not sufficient to dissociate NF-xcexaB from the latent complex (Palombella, V. J., Cell. 78:773-785 (1994); Traenckner, E. B.-M. et al., EMBO J. 13:5433-56441 (1994); Finco, T. S. et al., Proc. Natl. Acad. Sci. USA 91:11884-11888 (1994); Miyamoto, S. et al., Proc. Natl. Acad. Sci. USA 91:12740-12744 (1994); Lin, Y.-C. et al., Proc. Natl. Acad. Sci. USA 92:552-556 (1995); Alkalay, I. et al., Mol. Cell. Biol. 15:1294-1304 (1995); DiDonato, J. A. et al., Mol. Cell. Biol. 15:1302-1311 (1995)). Rather, phosphorylation triggers the degradation of IxcexaBxcex1 (Brown, K. et al., Science 267:1485-1491 (1995); Brockman, J. A. et al., Mol. Cell. Biol. 15:2809-2818 (1995); Traenckner, E. B.-M. et al., EMBO J. 14:2876-2883 (1995); Whiteside, S. T. et al., Mol. Cell. Biol. 15:5339-5345 (1995)).
Recently, it has been shown that signal-induced degradation of IxcexaBxcex1 is mediated by the ubiquitin-proteasome pathway (Chen, Z. J. et al., Genes and Dev. 9:1586-1597 (1995); Scherer, D. C. et al., Proc. Natl. Acad. Sci. USA 92:11259-11263 (1995); Alkalay, I. et al., Proc. Natl. Acad. Sci. USA. 92:10599-10603 (1995)). In this pathway, a protein targeted for degradation is first modified by covalent attachment of ubiquitin, a highly conserved polypeptide of 76 amino acids (reviewed by Hershko and Ciechanover, Annu. Rev. Biochem. 61:761-807 (1992); Ciechanover, A., Cell 79:13-21 (1994)). Ubiquitination is a three-step process: First, ubiquitin is activated by a ubiquitin activating enzyme (E1); the activated ubiquitin is then transferred to a ubiquitin carrier protein (E2, also referred to as ubiquitin conjugating enzyme or UBC); finally, ubiquitin is conjugated to a protein substrate by forming an isopeptide bond between the carboxyl terminal glycine residue of ubiquitin and the xcex5-amino group of one or more lysine residues of the protein substrate. This conjugation step often requires a ubiquitin protein ligase (E3). Multiple molecules of ubiquitin can be ligated to a protein substrate to form multi-ubiquitin chains, which are then recognized by a large, ATP-dependent protease (MW xcx9c2000 kDa) called the 26S proteasome. The 26S proteasome is composed of a 20S catalytic core, and a 19S regulatory complex (reviewed by Goldberg, A. L., Science 268:522-523 (1995)).
Multiple E2s and E3s function together to mediate the ubiquitination of a variety of cellular proteins. For example, there are at least a dozen E2s in yeast that display distinct substrate specificities and carry out distinct cellular functions. The closely related E2 proteins UBC4 and UBC5 are involved in the turnover of many short-lived and abnormal proteins, and they play an essential role in the stress response (Seufert and Jentsch, EMBO J. 9:543-550 (1990)). Homologs of UBC4/UBC5 mediate the ubiquitination of the P53 protein in conjunction with the HPV-16 E6-E6AP complex, which functions as an E3 (Schaffner, M. et al., Cell. 75:495-505 (1993)). These E2s have also been implicated in the ubiquitination of the MATxcex12 Processor (Chen, P. et al., Cell 74:357-369 (1993)), cyclin B (King, R. W. et al., Cell 81:279-288 (1995)), and the NF-xcexaB precursor protein P105 (Orian, A. et al., J. Biol. Chem. 270:21707-21714 (1995)). The involvement of UBC4/UBC5 in the ubiquitination of such diverse substrates indicates that these E2s alone cannot confer substrate specificity. However, they may act together with specific E3s to recognize specific substrates. Although relatively few E3s have been identified thus far, the existence of a large family of these proteins is likely (Huibregtse, J. M. et al., Proc. Natl. Acad. Sci. USA 92:2563-2567 (1995)).
Ubiquitination of IxcexaBxcex1 is regulated by signal-induced phosphorylation at two specific residues, serines 32 and 36 (Chen, Z. J. et al., Genes and Dev. 9:1586-1597 (1995)). Single amino acid substitutions of one or both of these residues abolish the signal-induced phosphorylation and degradation of IxcexaBxcex1 (Brown, K. et al., Science 267:1485-1491 (1995); Brockman, J. A. et al., Mol. Cell Biol. 15:2809-2818 (1995); Traenckner, E. B.-M. et al., EMBO J. 14:2876-2883 (1995); Whiteside, S. T. et al., Mol. Cell. Biol. 15:5339-5345 (1995)). The same mutations also abolish the okadaic acid-induced phosphorylation and ubiquitination of IxcexaBxcex1 in vitro (Chen, Z. J. et al., Genes and Dev. 9:1586-1597 (1995)). Relatively little is known about the signal transduction pathways and the kinase(s) responsible for the site-specific phosphorylation of IxcexaBxcex1. Mutants of IxcexaBxcex1 lacking serines 32 and 36 are resistant to induced phosphorylation by a variety of stimuli, suggesting that different signal transduction pathways converge on a specific kinase or kinases. However, despite considerable effort, the identification of this IxcexaBxcex1 kinase has remained elusive (Verma, I. M. et al., Genes and Dev. 9:2723-2735 (1995)).
Several serine/threonine kinases, including protein kinase C (PKC), heme-regulated eIF-2xcex1 kinase (HRI), protein kinase A (Ghosh and Baltimore, Nature 344:678-682 (1990)), casein kinase II (Barroga, C. F. et al., Proc. Natl. Acad. Sci. USA 92:7637-7641 (1995)) and a recently described 42 kDa kinase (Kano, K. et al., J. Biol. Chem. 270:27914-27919 (1995)), have been shown to phosphorylate IxcexaBxcex1 in vitro. However, none of these kinases have been shown to phosphorylate IxcexaBxcex1 at serines 32 and 36. Various kinases have also been implicated in the regulation of NF-xcexaB in vivo, such as xcex6PKC (Dominguez, I. et al., Mol. Cell. Biol. 13:1290-1295 (1993); Diaz-Meco, M. T. et al., EMBO J. 13:2842-2848 (1994)), ceramide-dependent protein kinase (Schutze, S. et al., Cell 71:765-776 (1992)), tyrosine kinases (Devary, Y. et al., Science 261:1442-1445 (1993)), Raf (Finco and Baldwin, J. Biol. Chem. 24:17676-17679 (1993); Li and Sedivy, Proc. Natl. Acad. Sci. USA 90:9247-9251 (1993)), and the Drosophila pelle kinase, which is required for the inactivation of cactus, a Drosophila IxcexaB homolog (Wassermann, S. A., Mol. Biol. Cell 4:767-771 (1993)). These kinases may function at various steps in the signal transduction pathway upstream of IxcexaBxcex1 phosphorylation, but none have been shown to directly phosphorylate IxcexaBxcex1 at relevant sites.
Although the diverse nature of NF-xcexaB stimuli suggests that the initial steps in the signal transduction pathways are distinct, these pathways appear to converge on the generation of reactive oxygen intermediates (ROIs, such as H2O2), which are thought to function as common second messenger-like molecules in the activation of NF-xcexaB (Schreck, R. et al., EMBO J. 10:2247-2258 (1991)). At present the mechanistic link between ROIs and IxcexaBxcex1 phosphorylation is not understood.
The establishment of an in vitro system for signal-induced phosphorylation and ubiquitination of IxcexaBxcex1 was previously reported (Chen, Z. J. et al., Genes and Dev. 9:1586-1597 (1995)). The present invention involves fractionating HeLa cell cytoplasmic extracts and assaying for specific phosphorylation and ubiquitination of IxcexaBxcex1. The present invention provides a high molecular weight kinase complex that, in its activated state, specifically phosphorylates IxcexaBxcex1 at serines 32 and 36. Surprisingly, UBC4/UBC5, ubiquitin, and E1 are not only involved in the ubiquitination of IxcexaBxcex1, but may also be required for the phosphorylation of IxcexaBxcex1. Additional experiments reveal that this IxcexaBxcex1 kinase can be activated by a prior ubiquitination event. In this case, ubiquitination serves a regulatory function without involving proteolysis. Additionally, this IxcexaBxcex1 may be activated by MEKK1.
Signal-induced activation of the transcription factor NF-xcexaB requires specific phosphorylation of the inhibitor IxcexaBxcex1 (SEQ ID NO:9) and its subsequent proteolytic degradation. Phosphorylation of serine residues 32 and 36 targets IxcexaBxcex1 to the ubiquitin-proteasome pathway. The present invention provides a substantially purified large, multi-subunit kinase (MWxcx9c700 kDa) that, in its active state, phosphorylates IxcexaBxcex1 at serines 32 and 36. Preferably, the kinase comprises an amino acid sequence which is at least 60% homologous to the amino acid sequence of any one of FIGS. 21A-D. Remarkably, this kinase may be activated by a ubiquitination event requiring the ubiquitin activating enzyme (E1), a specific ubiquitin carrier protein (E2) of the UBC4/UBC5 family, and ubiquitin. Thus, in this case, ubiquitination serves a novel regulatory function that does not involve proteolysis. Alternatively, the kinase may be activated via phosphorylation by MEKK-1. Additional activation routes, e.g. phosphorylation by a kinase other than MEKK-1, may also be possible.
The invention further provides the substantially pure polypeptide subunits of the above-described kinase.
The invention provides isolated nucleic acid molecules coding for subunits of the above-described kinase.
The invention also provides a nucleic acid probe for the specific detection of the presence of nucleic acid encoding the above-described kinase or its subunits or a fragment thereof in a sample.
The invention further provides a method of detecting the above-described nucleic acid in a sample.
The invention also provides a kit for detecting the presence of the above-described nucleic acid in a sample.
The invention further provides a recombinant nucleic acid molecule comprising, 5xe2x80x2 to 3xe2x80x2, a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecule.
The invention also provides a recombinant nucleic acid molecule comprising a vector and the above-described nucleic acid molecule.
The invention also provides a cell that contains the above-described recombinant nucleic acid molecule.
The invention further provides a non-human organism that contains the above-described recombinant nucleic acid molecule.
The invention also provides an antibody having binding affinity specifically to the above-described kinase or to a subunit of the above-described kinase.
The invention further provides a method of detecting the above-described kinase or one of its subunits in a sample.
The invention also provides a method of measuring the amount of the above-described kinase in a sample.
The invention further provides a diagnostic kit comprising a first container means containing the above-described antibody, and a second container means containing a conjugate comprising a binding partner of the monoclonal antibody and a label.
The invention also provides a hybridoma which produces the above-described monoclonal antibody.
The invention further provides diagnostic methods for human disease, in particular diseases, disorders, and injuries resulting from an undesired activation of NF-xcexaB.
The invention also provides methods for therapeutic uses involving (1) the nucleic acid sequence encoding the above-described kinase or a subunit thereof and/or (2) the above-described kinase or a subunit thereof.
The invention provides ligands, agonists, and antagonists of the above-described kinase and diagnostic and therapeutic uses for these molecules. Preferably, the molecule is a selective inhibitor of kinase activity, i.e. the ability to phosphorylate IxcexaBxcex1 at serine residues 32 and 36.
The invention also provides assays for the identification of ligands, agonists and antagonists of the above-described kinase.
Further objects and advantages of the present invention will be clear from the description that follows.
In the description that follows, a number of terms used in recombinant DNA (rDNA) technology, protein purification, and diagnostic and therapeutic methods are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Isolated Nucleic Acid Molecule. An xe2x80x9cisolated nucleic acid moleculexe2x80x9d, as is generally understood and used herein, refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA.
DNA Segment. A DNA segment, as is generally understood and used herein, refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that can encode, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.
Gene. A DNA sequence related to a single polypeptide chain or protein, and as used herein includes the 5xe2x80x2 and 3xe2x80x2 untranslated ends. The polypeptide can be encoded by a full-length sequence or any portion of the coding sequence, so long as the functional activity of the protein is retained.
Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesized by reverse transcription of messenger RNA (xe2x80x9cmRNAxe2x80x9d).
Structural Gene. A DNA sequence that is transcribed into mRNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
Restriction Endonuclease. A restriction endonuclease (also restriction enzyme) is an enzyme that has the capacity to recognize a specific base sequence (usually 4, 5, or 6 base pairs in length) in a DNA molecule, and to cleave the DNA molecule at every place where this sequence appears. For example, EcoRI recognizes the base sequence GAATTC/CTTAAG.
Restriction Fragment. The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome can be digested by a particular restriction endonuclease into a discrete set of restriction fragments.
Polyacrylamide Gel Electrophoresis (PAGE). The most commonly used technique (though not the only one) for achieving a fractionation of polypeptides on the basis of size is polyacrylamide gel electrophoresis. The principle of this method is that polypeptide molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. Note that the smaller the polypeptide fragment, the greater the mobility under electrophoresis in the polyacrylamide gel. Both before and during electrophoresis, the polypeptides typically are continuously exposed to the detergent sodium dodecyl sulfate (SDS), under which conditions the polypeptides are denatured. Native gels are run in the absence of SDS.
The polypeptides fractionated by polyacrylamide gel electrophoresis can be visualized directly by a staining procedure if the number of polypeptide components is small.
Western Transfer Procedure. The purpose of the Western transfer procedure (also referred to as blotting) is to physically transfer polypeptides fractionated by polyacrylamide gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of polypeptides resulting from the fractionation procedure. The blot is then probed with an antibody that specifically binds to the polypeptide of interest.
Nucleic Acid Hybridization. Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern hybridization procedure, the latter situation occurs. As noted previously, the DNA of the individual to be tested is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single-stranded form, and transferred to nitrocellulose paper, making it available for reannealing to the hybridization probe.
Antibody Probe. To visualize a particular polypeptide sequence in the western blot procedure, a labeled antibody probe is exposed to the fractionated polypeptides bound to the nitrocellulose filter. The areas on the filter that carry polypeptides that bind to the labeled antibody probe become labeled themselves as a consequence of the binding. The areas of the filter that exhibit such labeling are visualized.
Stringent Hybridization Conditions. Examples of hybridization conditions can be found in Ausubel, F. M. et al., Current protocols in Molecular Biology, John Wily and Sons, Inc., New York, N.Y. (1989). A nitrocellulose filter is incubated overnight at 68xc2x0 C. with labeled probe in a solution containing 50% formamide, high salt (either 5xc3x97 SSC[20xc3x97: 3M NaCl/0.3M trisodium citrate] or 5xc3x97 SSPE [20xc3x97: 3.6M NaCl/0.2M NaH2PO4/0.02M EDTA, pH 7.7]), 5xc3x97 Denhardt""s solution, 1% SDS, and 100 xcexcg/ml denatured salmon sperm DNA. This is followed by several washes in 0.2xc3x97 SSC/0.1% SDS at a temperature selected based on the desired stringency: room temperature (low stringency), 42xc2x0 C. (moderate stringency) or 68xc2x0 C. (high stringency). The temperature selected is determined based on the melting temperature (Tm) of the DNA hybrid.
Oligonucleotide or Oligomer. A molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. An oligonucleotide can be derived synthetically or by cloning.
Sequence Amplification. A method for generating large amounts of a target sequence. In general, one or more amplification primers are annealed to a nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to, or in between the primers are amplified.
Amplification Primer. An oligonucleotide which is capable of annealing adjacent to a target sequence and serving as an initiation point for DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated.
Vector. A plasmid or phage DNA or other DNA sequence into which DNA can be inserted to be cloned. The vector can replicate autonomously in a host cell, and can be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences can be cut in a determinable fashion and into which DNA can be inserted. The vector can further contain a marker suitable for use in the identification of cells transformed with the vector. Markers, for example, are tetracycline resistance or ampicillin resistance. The words xe2x80x9ccloning vehiclexe2x80x9d are sometimes used for xe2x80x9cvector.xe2x80x9d
Expression. Expression is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s).
Expression Vector. A vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
Functional Derivative. A xe2x80x9cfunctional derivativexe2x80x9d of a sequence, either protein or nucleic acid, is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the protein or nucleic acid sequence. A functional derivative of a protein can contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term xe2x80x9cfunctional derivativexe2x80x9d is intended to include the xe2x80x9cfragments,xe2x80x9d xe2x80x9csegments,xe2x80x9d xe2x80x9cvariants,xe2x80x9d xe2x80x9canalogs,xe2x80x9d or xe2x80x9cchemical derivativesxe2x80x9d of a molecule.
As used herein, a molecule is said to be a xe2x80x9cchemical derivativexe2x80x9d of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule""s solubility, absorption, biological half life, and the like. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington""s Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.
Variant. A xe2x80x9cvariantxe2x80x9d of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid. Thus, provided that two molecules possess a common activity and can substitute for each other, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
Allele. An xe2x80x9callelexe2x80x9d is an alternative form of a gene occupying a given locus on the chromosome.
Mutation. A xe2x80x9cmutationxe2x80x9d is any detectable change in the genetic material which can be transmitted to daughter cells and possibly even to succeeding generations giving rise to mutant cells or mutant individuals. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or area of cells arises. Mutations in the germ line of sexually reproducing organisms can be transmitted by the gametes to the next generation resulting in an individual with the new mutant condition in both its somatic and germ cells. A mutation can be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides can be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations can occur spontaneously and can be induced experimentally by application of mutagens or by site-directed mutagenesis. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide can result from a mutant nucleic acid molecule.
Species. A xe2x80x9cspeciesxe2x80x9d is a group of actually or potentially interbreeding natural populations. A species variation within a nucleic acid molecule or protein is a change in the nucleic acid or amino acid sequence that occurs among species and can be determined by DNA sequencing of the molecule in question.
Purified. A xe2x80x9cpurifiedxe2x80x9d protein or nucleic acid is a protein or nucleic acid preparation that is generally free of contaminants, whether produced recombinantly, chemically synthesized or purified from a natural source.
% Homologous. When referring to one amino acid sequence as being X% homologous to another amino acid sequence, what is meant is the percentage of sequence identity or sequence similarity. Amino acid sequence similarity is further described in Table 1, infra.
Subunit of the Kinase. The kinase is a multi-subunit protein. Each subunit is defined herein to be a single polypeptide which is encoded by a nucleic acid sequence.
p85, p70, p62, p55, p50, p43, p40, p38, p36, p33, p31.
For purposes of the invention, these terms refer to polypeptide subunits of the above-described kinase, wherein each subunit has a corresponding molecular weight observed by SDS PAGE. For example, p85 is a polypeptide subunit with approximate molecular weight of 85 KDa observed by SDS PAGE, of a large, multisubunit kinase that in its active state phosphorylates IxcexaBxcex1 at serines 32 and 36. p70 has an approximate molecular weight by SDS PAGE of 70 KDa, p62 has an approximate molecular weight by SDS PAGE of 62 kDa, and so on.
Substrate. A substrate for the kinase is a ligand which becomes phosphorylated as a result of its interaction with the kinase.
Ligand. Ligand refers to any molecule that can interact with the above-described kinase or a subunit thereof. The ligand can be a naturally occurring polypeptide, or may be synthetically or recombinantly produced. The ligand can be soluble or membrane bound. The ligand can also be a nonprotein molecule that acts as a ligand when it interacts with the kinase. Interactions between the ligand and the kinase include, but are not limited to, any covalent or non-covalent interactions. Preferably, the ligand interacts selectively with the kinase. Agonists and antagonists of the kinase that can interact with the kinase are examples of ligands according to the present invention. Preferably, the ligand is a selective inhibitor of the kinase activity, i.e. the ability to phosphorylate IxcexaB-xcex1 at serine residues 32 and 36.
Disease states characterized by undesired activation of NF-xcexaB. The phrase disease states characterized by undesired activation of NF-xcexaB includes, but is not limited to, disease states in a mammal which can include inflammation, HIV infection, cancer, sepsis, psoriasis, and restenosis.
Drug. Drugs include, but are not limited to proteins, peptides, degenerate peptides, agents purified from conditioned cell medium, organic molecules, inorganic molecules, antibodies or oligonucleotides. Other candidate drugs include analogs of the above-described kinase ligand or ligands. The drug can be naturally occurring or synthetically or recombinantly produced. One skilled in the art will understand that such drugs can be developed by the assays described below.